Sialic acid residues on astrocytes regulate neuritogenesis by controlling the assembly of laminin matrices

Abstract

In the developing nervous system migrating neurons and growing axons are guided by diffusible and/or substrate-bound cues, such as extracellular matrix-associated laminin. In a previous work we demonstrated that laminin molecules could self-assemble in two different manners, giving rise to matrices that could favor either neuritogenesis or proliferation of cortical precursor cells. We investigated whether the ability of astrocytes to promote neuritogenesis of co-cultivated neurons was modulated by the assembling mode of the laminin matrix secreted by them. We compared the morphologies and neuritogenic potentials of laminin deposited by in vitro-differentiated astrocytes obtained from embryonic or neonatal rat brain cortices. We showed that, while permissive astrocytes derived from embryonic brain produced a flat laminin matrix that remained associated to the cell surface, astrocytes derived from newborn brain secreted a laminin matrix resembling a fibrillar web that protruded from the cell plane. The average neurite lengths obtained for E16 neurons cultured on each astrocyte layer were 198±22 and 123±13 μm, respectively. Analyses of surface-associated electrostatic potentials revealed that embryonic astrocytes presented a pI of -2.8, while in newborn cells this value was -3.8. Removal of the sialic acid groups on the embryonic monolayer by neuraminidase treatment led to the immediate release of matrix-associated laminin. Interestingly, laminin reassembled 1 hour after neuraminidase removal converted to the features of the newborn matrix. Alternatively, treatment of astrocytes with the cholesterol-solubilizing detergent methyl-β-cyclodextrin also resulted in release of the extracellular laminin. To test the hypothesis that sialic-acid-containing lipids localized at cholesterol-rich membrane domains could affect the process of laminin assembly, we devised a cell-free assay where laminin polymerization was carried out over artificial lipid films. Films of either a mixture of gangliosides or pure ganglioside GT1b induced formation of matrices of morpho-functional features similar to the matrices deposited by embryonic astrocytes. Conversely, films of phosphatidylcholine or ganglioside GM1 led to the formation of bulky laminin aggregates that lacked a defined structure. We propose that the expression of negative lipids on astrocytes can control the extracellular polymerization of laminin and, consequently, the permissivity to neuritogenesis of astrocytes during development.

Artificial Laminin Polymers Assembled in Acidic pH Mimic Basement Membrane Organization

“…Deposited on Coverslip-Although laminin aggregates could not be detected in neutral pH at 50 g/ml, we have previously demonstrated that deposits of the aggregated protein became visible when the laminin solution was incubated over the surface of a glass coverslip (11,14). Such deposits were characterized by immunostaining and shown to be morphologically different from others obtained in acidic pH.…”

“…In the nervous system, for instance, it has been demonstrated that laminin goes from cell-associated to scattered extracellular matrix deposits or from a punctated pattern to small or large sheet-like aggregates in developing brain (4,12). Cells in culture can also assemble different networks as demonstrated for midbrain and cortical neural cells (13,14) or Schwann cells (15). In addition, we have previously described that the laminin matrix synthesized by embryonic astrocytes (which efficiently supports neurite outgrowth) resembles the artificial matrix assembled upon laminin acidification because it is flat, organized, and closely associated to the cell surface (14).…”

“…Cells in culture can also assemble different networks as demonstrated for midbrain and cortical neural cells (13,14) or Schwann cells (15). In addition, we have previously described that the laminin matrix synthesized by embryonic astrocytes (which efficiently supports neurite outgrowth) resembles the artificial matrix assembled upon laminin acidification because it is flat, organized, and closely associated to the cell surface (14).…”

Lysophosphatidic Acid Receptor-dependent Secondary Effects via Astrocytes Promote Neuronal Differentiation

“…Astrocytes, the most abundant glial cell type, produce most of the extracellular matrix components and neurotrophic factors in the central nervous system that are involved in axonal growth (18,19,22,32), neuronal proliferation, survival, and stem cell fate determination (14,19,20,26,(33)(34)(35)(36)(37).…”

Heparin-binding Sites in Granulocyte-Macrophage Colony-stimulating Factor

“…For example, the inhibition of nerve regeneration mediated by myelin-associated glycoprotein has been shown to involve clustering of gangliosides (43). In another model, the assembly of laminin on the surface of astrocytes in culture as well as the laminin-induced neuritogenesis of primary neurons cultivated on them have been shown to be controlled by the local acidic pH provided by sialic acid-containing lipid rafts on the membranes of astrocytes (44,45).…”